It is known that to obtain optimal yields in bioreactors and fermentors active monitoring and control of basic environmental factors is necessary [James Lee, Biochemical Engineering, Washington State University, e-book, 2002]. The most critical of these factors include temperature, dissolved oxygen level, and pH. The dominant paradigm for monitoring these quantities in stainless steel or glass tank type bioreactors and fermentors has been through the use of electrochemical probes. Despite some of the drawbacks associated with this type of probe, they have proven to be acceptable in performance level and currently enjoy widespread use in glass and steel bioreactors/fermentors.
However, recent years have seen the rise in popularity of disposable bioreactors fabricated of bio-compatible polymers to supplement, or in many cases replace glass and stainless steel bioreactors. A major issue has been the ability to continuously and reliably provide on-line monitoring of pH and dissolved oxygen for these disposable (polymeric) bioreactor vessels. Part of the issue is that it has been difficult to successfully implement electrochemical probes with disposable bioreactors. The main issues with the use of electrochemical probes are three fold:
1. Size;
2. Electrical grounding issues.
3. Inability maintain a sterile system.
The majority of disposable bioreactors currently sold are comprised of flexible, biocompatible (USP, FDA regulation compliant, and animal product derived free) polymers. There is often difficulty in mounting rigid glass and steel probes to the polymeric bioreactor surfaces which flex dynamically in operation. Methods to circumvent this problem have been attempted, but inserting the probes into disposable bioreactors while maintaining the sterility and integrity of the seal remains problematic, especially for smaller bag reactors. Additionally, electrochemical probes are prone to grounding issues and electrical ingress noise when used with a polymeric, non-conductive, disposable bioreactor. In a traditional stainless steel bioreactor, these factors are typically not a concern as the probes are in contact with the metal wall of the bioreactor, which can be grounded. However, when the bioreactor is made solely or predominantly of a flexible, dielectric (polymeric) material such as low density polyethylene, polypropylene, or ethylene-vinyl acetate (EVA) it is difficult to provide the requisite shielding and grounding. Most importantly, much of the appeal of single use bioreactors is the ability to eliminate time consuming work and capital infrastructure associated with sterilization, and cleaning as compared to a traditional metal or glass bioreactor. Users of disposable bioreactors would strongly prefer to receive a pre-sterilized (typically with Gamma or Beta radiation) bioreactor with calibrated probes in place and filled with media and simply start their process. Inserting anything into the already sterilized disposable bag reactor adds another layer of uncertainty and effort. We have found that optical technology based probes can address many of the aforementioned issues.
In recent years, disposable bioreactors have proliferated, and can now be found in many shapes and sizes. Some of the incipient work in making the disposable bioreactor a commercial reality was done by Wavetech. Their bioreactor is based on a disposable bag (often referred to as a rocker bag or a pillow bag) made of polyethylene and/or other suitable biocompatible plastic. The bag sits on a device that rocks the bag back and forth to both mix and oxygenate the contents. The espoused theory is that this motion is similar to waves in the ocean and is therefore beneficial to water based life in general. (See U.S. Pat. No. 6,190,913, Singh, Vijay, Method for Culturing Cells Using Wave-induced Agitation). A similar approach was taken by Applikon for its Appliflex product, and Metabios for its Optima product. Hyclone and Xcellerex have developed a different style of disposable bioreactor based on a disposable liner which sits inside a rigid container vessel and seeks to mimic the behavior of a traditional bioreactor. Specifically, a stainless steel container, or other rigid structure that serves as external support, is lined with a disposable biocompatible plastic membrane and uses a traditional impeller mixing system.
A method of monitoring critical bioprocess parameters such as dissolved oxygen and pH that addresses some of the existing issues with disposable bioreactors is provided by the use of fluorescent optical sensor materials [Wolbeis, O. S., Fiber Optic Chemical Sensors and Biosensors, Vol 1&2 CRC, Boca Raton, 1991.]. These fluorescent optical sensor materials operate on the principle of dynamic quenching [Lakowicz, Principles of Fluorescence of Spectroscopy, 3rd edition, Springer 2006]. The currently preferred term for these materials is fluorescent dye, indicator dye or fluorophore and such terms will be used interchangeably hereinafter. The fluorescent lifetime of substances such as organo-metallic fluorophores or Pt metal group based fluorophores is quenched, or shortened, by contact with the analyte under study. Dissolved oxygen is a natural analyte to measure using this method, as oxygen is known to readily quench the fluorescence of many fluorescent dyes. However, in addition to O2 fluorophores suitable for the detection of other analytes such as, CO2, pH and glucose are known in the art. Therefore, pH, CO2, and glucose sensors can be constructed using this technology and suitable fluorophores. Despite the fact that the fluorescence quenching effect was discovered by Kautsky in 1939 [Hans Kautsky, Quenching of Luminescence by Oxygen, Trans. Faraday Soc., 1939, 35, 216-219] it has taken more than 60 years for this technology to evolve to a level where it is suitable for biotechnology based instrumentation.
Another similar area of interest for use in monitoring bioreactors is the use of auto-fluorescence. In some instances, it is desireable to use visible and ultra-violet wavelength excitation to exploit naturally occurring fluorescence behavior to detect the presence of analytes or cell characteristics of interest. For example, many known metabolic markers such as NADH, FAD, and tryptophan, are known to auto-fluoresce and can be utilized in accordance with the teaching of the present invention to give information regarding cell viability and cell energy.
Despite the promise of this optical technology, it faces a fundamental issue due to the fact that many indicator dyes (fluorophores), especially those based on porphryins, tend to photo-degrade. This photo-degradation is often termed static quenching and is due to the fact that in their excited states fluorophores often react to form the photo-stable ground states of other non-fluorescing compounds. This behavior causes a fundamental change in the optical response characteristics of the fluorescent dye and can lead to a loss in signal level and to calibration error or signal drift. Significant effort has been put forth in an effort to understanding the degradation mechanisms, and how to mitigate them. Several strategies have been suggested [P. S. Dittrich and P. Schwille, Photobleaching and stabilization of fluorophores used for single-molecule analysis with one and two photon excitation, Applied Physics B 73, 829-837, 2001, Sandra Bencic-Nagale and David R Walt, Extending the Longevity of Fluorescence-Based Sensor Arrays Using Adaptive Exposure, Anal, Chem., 77, 6155-6162, 2005 However, the most straight forward solution is to limit the total fluence (energy×time) or photon exposure to which the photo-sensitive fluorophores are subjected. This solution is achieved by the present invention and is addressed in detail hereinafter.
The common thread among all existing types of disposable bioreactors is that, as previously indicated, they utilize a biocompatible dielectric material which is intended to be discarded after a single use. These biocompatible materials are generally translucent, but not transparent in the visible region of the optical spectrum. In fact, reactions between room light and the dielectric materials have motivated many vendors to provide more or less opaque covers for the bioreactor. The basic point is that the transmission of light through these disposable products is not a property specifically desired or engineered into them. Additionally, disposable bioreactors are often constructed as a composite, multi-layer structure in which an inner layer comprising polyethylene, polypropylene, or EVA, is combined with an outer layer comprising nylon, Teflon, or another material depending on whether strength, oxygen permeability or other specific characteristic is most desired. Irrespective of the precise reason for the layering, the end result is that the optical transmission properties of the bag are significantly compromised. The importance of this issue to the implementation of fluorescent optical sensors will be discussed shortly, but it is necessary to first review some of the basic principles which govern the operation of fluorescence based optical sensors.
The dynamic quenching of fluorescence is described to first order by the Stern Volmer equations:
                                                        l              0                        l                    =                      1            +                                          K                SV                            ⁢              p              ⁢                                                          ⁢                              O                2                                                    ⁢                                  ⁢                                            τ              0                        τ                    =                      1            +                                          K                SV                            ⁢              p              ⁢                                                          ⁢                              O                2                                                    ⁢                                  ⁢                              K            sv                    =                      k            ⁢                                                  ⁢                          τ              0                                                          Equations        ⁢                                  ⁢        1            Where I is the fluorescence intensity, I0 is the fluorescence intensity in the absence of oxygen, τ is the fluorescent lifetime, and τ0 is the fluorescent lifetime in the absence of oxygen, KSV is the Stern Volmer constant, and k is the bi-molecular quenching constant.
These equations and the physical process they describe are the basis for how these sensors indicate the change in concentration of the species (i.e., analyte, quencher) under study. Using this principle it is possible to sense the change in intensity or lifetime of the fluorophore which comes in contact with the quencher species of interest. For example, in the absence of the quencher the lifetime of the fluorophore will be at its longest and the fluorescent intensity therefore at its highest. As the concentration of the quencher increases, both the fluorescent lifetime and the fluorescent intensity decay. For a number of reasons, including cost and simplicity, sensing the change in the lifetime in the frequency domain is the dominant methodology for current commercial instrumentation. In contradistinction, in the implementation of the present invention, the change in lifetime is sensed through a change in the phase delay of the fluorescent radiation, as compared to the excitation emission [See J. Lakowicz, Principles of Fluorescence Spectroscopy]. A light source, at a wavelength at which the fluorophore (frequently, but not necessarily an organo-metallic compound) absorbs, is modulated and the emitted light is monitored. The emitted light is typically analyzed using phase sensitive detection systems such as a lock-in amplifier to examine the phase delay between the excitation source and the emitted radiation.
In the simplest case, this change in phase can be expressed as:φ=ArcTan(2πfτ)  Eq. 2Where f is the modulation frequency of the excitation source, and τ is lifetime of the fluorophore as described by the Stern Volmer equations. (Equations 1)
A prior art approach for implementing the aforementioned phase delay analysis is shown in FIG. 1. This design uses fiber optic delivery of the excitation light, as well as fiber based collection of the fluorescence signal.
In FIG. 1, 1 is the excitation light such as an LED, which is normally sinusoidally modulated, 2 is an optical filter which tailors the optical excitation spectrum such that it is matched to the absorption feature of the fluorophore 6. This absorption feature, and hence the excitation spectrum will vary depending on the exact fluorophore used and the matrix in which it is complexed. In general, it is advantageous to use the longest wavelength (lowest energy) photons that will excite the fluorophore. Component 3 is a fiber optic coupler which allows the excitation light to travel to the common delivery/collection fiber 4, while allowing the fluorescent signal to simultaneously travel in the opposite direction. This fluorescent signal passes through filter 7 which is designed such that only the fluorescent signal reaches the optical detector 8; i.e., any pump light or ambient light is blocked from reaching the detector. An optional set of coupling optics is shown in the design of FIG. 9 which helps increase collection and delivery of light from and to the fluorophore, respectively.
A key issue in fluorescent optical probes is the trade-off between the signal-to-noise ratio (“SNR”) and photo-degradation of the fluorophore. The higher the excitation light power level the better the signal to noise ratio, but the more rapid the photo-degradation rate of the fluorophore. It therefore follows that effective collection of the fluorescent photons can play a big part in controlling the photo-degradation rate. Specifically, the more efficiently the fluorescence is collected, the higher the SNR and therefore the lower the required intensity of the excitation light. Likewise, the lower the intensity of the excitation light source, the slower the photo-degradation rate. This is important, because in fiber optic based systems of the prior art as described above, a large percentage of the fluorescent signal light is not utilized due to the etendue (as described below) limitations of optical fiber based designs.
In the branch of optics known as radiometry, the concept of brightness or etendue is very important because it is a conserved quantity. Etendue is expressed as:S=AΩ  Eq. 3Where S represents the etendue, A is area of the light source or detector, and Ω is the solid angle that the source emits into, or that it is collected by the detector.
Brightness is the optical power divided by the etendue and this is a fundamental conserved quantity. It is therefore impossible to increase the brightness of a source using passive optical elements [See Ross McCluney, Introduction to Radiometry and Photometry, Artech House, 1994]. The relevant issue for fluorescent optical sensors, and in particular, fiber optic based fluorescent optical sensors is that due to the limited modal area of the fiber and its limited acceptance angle or numerical aperture the etendue is fundamentally limited.
In a typical plastic optical fiber used for the collection of light, the mode field diameter can be as large as 1 mm with a numerical aperture of 0.63. A numerical aperture of 0.63 means that the half angle of acceptance of the fiber is Arcsine (0.63) i.e., approximately 39 degrees—or a full angle of 78 degrees out of the total of 180 degrees in the half plane. However, the fluorescent material is typically in the form of a thin disk or dot which is 3-5 mm in diameter and emits into all space with Lambertian characteristics (e.g., an intensity that has a Cos(θ) dependence and where θ is the angle between the normal to the disk and the angle of emission of the photon). Conservation of brightness tells us that therefore only a very small percentage of the emitted fluorescent light can be collected by the fiber. Since it is difficult to integrally and seamlessly integrate optically transparent materials to the plastic bioreactor bag further loss can occur.
1. The fiber tip is not located very close to the fluorescent material or if the fluorescent material is located on the other side of a diffusive material (e.g. a multi-layer bag, or a textured bag material); and/or
2. If the distance and orientation of the emitter and receiving fiber is not fixed. Therefore, either of these conditions will lead to further deterioration of the coupling efficiency.
The issues described above are typical of the issues encountered when fiber optic based delivery and collection systems are used with fluorescent sensors on disposable bioreactors. These issues can lead to decreased performance and reliability, while simultaneously increasing the photo-degradation rate of the fluorophore. Often the photo-degradation rate in fiber optic based designs is higher than other designs as the excitation intensity level is increased to make up for collection shortfalls. It is possible to mitigate some of these effects by either maximizing the light collection efficiency, and/or by using a larger area of fluorophore (i.e., a larger fluorescent dye spot) and simply generating and collecting more photons irrespective of the efficiency. Specifically, if the fluorescent dye spot is larger, then the illumination intensity can remain the same or perhaps even be lowered and the optical collection efficiency can remain the same, yet produce a higher fluorescent signal level. The brightness of the fluorescent source as described by equation 3 has not been improved, but a higher signal level is obtained at the cost of using a larger area. When using an optical fiber to receive and/or illuminate the fluorophore, the usable spot size is inherently limited by the fiber diameter.